1. Technical Field
The present invention relates to methods for enhancing the production of interferon in cell culture.
2. Background
Interferons (IFNs) can be classified into two major groups based on their primary sequence. Type I interferons, IFN-xcex1 and IFN-xcex2, are encoded by a super family of intron-less genes consisting of the IFN-xcex1 gene family and a single IFN-xcex2 gene. Type II interferon, or IFN-xcex3, consists of only a single type and is restricted to lymphocytes (T-cells and natural killer cells). Type I interferons mediate diverse biological processes including induction of antiviral activities, regulation of cellular growth and differentiation, and modulation of immune functions (Sen, G. C. and Lengyel, P. (1992) J. Biol. Chem. 267, 5017-5020; Pestka, S. and Langer, J. A. (1987) Ann. Rev. Biochem. 56, 727-777). The induced expression of Type I IFNs, which include the IFN-xcex1 and IFN-xcex2 gene families, is detected typically following viral infections. Many studies have identified promoter elements and transcription factors involved in regulating the expression of Type I IFNs (Du, W., Thanos, D. and Maniatis, T. (1993) Cell 74, 887-898; Matsuyama, T., Kimura, T., Kitagawa, M., Pfeffer, K., Kawakami, T., Watanabe, N., Kundig, T. M., Amakawa, R. Kishihara, K., Wakeham, A., Potter, J., Furlonger, C. L., Narendran, A., Suzuki, H., Ohashi, P. S., Paige, C. J., Taniguchi, T. and Mak, T. W. (1993) Cell 75, 83-97; Tanaka, N. and Taniguchi, T. (1992) Adv. Immunol. 52, 263-81). However, it remains unclear what are the biochemical cues that signify viral infections to the cell and the signaling mechanisms involved (for a recent review of the interferon system, see Jaramillo et al. Cancer Investigation 1995 13:327-337).
IFNs belong to a class of negative growth factors having the ability to inhibit growth of a wide variety of cells with both normal and transformed phenotypes. IFN therapy has been shown to be beneficial in the treatment of human malignancies such as Kaposi""s sarcoma, chronic myelogenous leukemia, non-Hodgkin""s lymphoma and hairy cell leukemia as well as the treatment of infectious diseases such as papilloma virus (genital warts) and hepatitis B and C (reviewed by Gutterman Proc. Natl Acad Sci. 91:1198-1205 1994). Recently genetically engineered, bacterially produced IFN-xcex2 was approved for treatment of multiple sclerosis, a relatively common neurological disease affecting at least 250,000 patients in the United States alone.
Currently, IFNs for therapeutic use are produced from two major sources, natural IFNs from human leukocytes or leukocyte cell lines and recombinant IFNs produced in bacterial cells. Natural IFNs are considered to be superior as they consist of the entire complement of IFNs and have the proper structure, but they are expensive and time-consuming to produce. Bacterially produced recombinant IFN is cheaper and more efficient to make, but studies have shown much higher rates of rejection for the bacterially produced protein, particularly after long term usage. For instance, previous medical studies have shown that the incidence of rejection as reflected by antibody formation can be as high as 20 to 38% for bacterially-produced IFN compared with only 1.2% for natural IFN-xcex1 (Antonelli et al. J. Inf. Disease 163:882-885 1991; Quesada et al. J. Clin. Oncology 3:1522-1528 1985). Thus, a method for enhancing the production of natural IFN to make it less expensive to produce would be advantageous.
IFNs elicit their biological activities by binding to their cognate receptors followed by signal transduction leading to induction of IFN-stimulated genes (ISGs). Some ISGs have been characterized and their biological activities examined. The best studied examples of ISGs include a double-stranded-RNA-dependent kinase (dsRNA-PKR, or just PKR, formerly known as p68 kinase), 2xe2x80x2-5xe2x80x2-linked oligoadenylate (2-5A) synthetase, and Mx proteins (Taylor J L, Grossberg S E. Virus Research 1990 15:1-26; Williams B R G. Eur. J. Biochem. 1991 200:1-11).
PKR (short for protein kinase, RNA-dependent) is the only identified dsRNA-binding protein known to possess a kinase activity. PKR is a serine/threonine kinase whose enzymatic activation required dsRNA binding and consequent autophosphorylation (Galabru, J. and Hovanessian, A. (1987) J. Biol. Chem. 262, 15538-15544; Meurs, E., Chong, K., Galabru, J., Thomas, N. S., Kerr, I. M., Williams, B. R. G. and Hovanessian, A. G. (1990) Cell 62, 379-390). PKR has also been referred to in the literature as dsRNA-activated protein kinase, P1/e1F2 kinase, DAI or dsI (for dsRNA-activated inhibitor), and p68 (human) or p65 (murine) kinase. Analogous enzymes have been described in rabbit reticulocytes, different murine tissues, and human peripheral blood mononuclear cells (Farrel et al. (1977) Cell 11, 187-200; Levin et al. (1978) Proc. Natl Acad. Sci. USA 75, 1121-1125; Hovanessian (1980) Biochimie 62, 775-778; Krust et al. (1982) Virology 120, 240-246; Buffet-Janvresse et al. (1986) J. Interferon Res. 6, 85-96). The best characterized in vivo substrate of PKR is the alpha subunit of eukaryotic initiation factor-2 (eIF-2a) which, once phosphorylated, leads ultimately to inhibition of cellular and viral protein synthesis (Hershey, J. W. B. (1991) Ann. Rev. Biochem. 60, 717-755). This particular function of PKR has been suggested as one of the mechanisms responsible for mediating the antiviral and antiproliferative activities of IFN-xcex1 and IFN-xcex2. An additional biological function for PKR is its putative role as a signal transducer. Kumar et al. demonstrated that PKR can phosphorylate IxcexaBxcex1, resulting in the release and activation of nuclear factor xcexaB (NF-xcexaB) (Kumar, A., Haque, J., Lacoste, J., Hiscott, J. and Williams, B. R. G. (1994) Proc. Natl. Acad. Sci. USA 91, 6288-6292). Given the well-characterized NF-xcexaB site in the IFN-xcex2 promoter, this may represent a mechanism through which PKR mediates dsRNA activation of IFN-xcex2 transcription (Visvanathan, K. V. and Goodbourne, S. (1989) EMBO J. 8, 1129-1138).
The present inventor have surprisingly discovered that manipulating the expression of certain ISGs can have beneficial uses in interferon production. They have discovered that over-expression of the PKR protein induces overproduction of the IFN-xcex1 and IFN-xcex2 interferons, which is useful for the enhanced production of interferon in animal cell culture.
3. Relevant Literature
Currently there are two major approaches to large-scale production of interferons: recombinant IFN produced in bacterial or mammalian cells or natural IFNs from human leukocyte cells following stimulation with viruses or other IFN inducers. U.S. Pat. No. 5,376,567 and U.S. Pat. No. 4,966,843 describe the production in Chinese hamster ovary cells of a recombinant human interferon; U.S. Pat. No. 5,196,323 describes the production of recombinant human IFN-xcex1 in E. coli cells. A number of patents describe the production of interferon from human leukocyte cells using a variety of protocols; for example, U.S. Pat. No. 4,745,053 describes a process for producing interferon from whole human blood using a viral inducer, U.S. Pat. No. 4,680,261 describes a process for inducing production of interferon in mammalian cell culture using an ascorbic acid derivative or an inorganic vanadium compound, and U.S. Pat. No. 4,548,900 describes a process for the induction of interferon using a polyhydric alcohol in a priming stage. The major disadvantage of the current methods of interferon production is that typically virus is used as the IFN inducer because other inducers do not produce high enough levels of interferon for most commercial purposes. The virus must then be removed from the interferon before use, which adds time and cost to the production method. In addition, use of virus as an inducer ultimately results in the death of the interferon-producing cells, so that no recycling and re-use of the cells is possible.
The present invention overcomes these problems by providing a interferon-production system that does not require the use of a viral inducer in order to achieve high levels of interferon production. Although viral inducers can be used with the systems of the invention, other inducers that do require removal prior to use of the IFNs are still capable of producing IFNs at commercially acceptable levels.
It is an object of this invention to provide a method for enhancing the production of interferon in animal cell culture without requiring the addition of virus as an inducer.
This object is generally accomplished by providing animal cell cultures in which the expression of the interferon genes is substantially increased from the normal level of expression. This may be effected by manipulating the level of expression of factors that function in vivo to regulate the interferon level, including interferon transcriptional regulators (for example, IRF1), interferon receptors, and interferon stimulated gene products (for example PKR and 2-5A synthetase).
Thus, increased production of INF and other objects of the invention as will hereinafter become apparent are accomplished by carrying out interferon production in animal cell cultures in which the level of interferon-regulating protein activity, particularly for double-stranded-RNA-dependent kinase (PKR) and 2xe2x80x2-5xe2x80x2 oligoadenylate synthetase (2-5A synthetase), is significantly increased from normal levels.